A hallmark feature of cancerous cells is uncontrolled proliferation. Among the differences that have been discovered between tumor and normal cells is resistance to the process of programmed cell death, also known as apoptosis (Ambrosini et al., Nat. Med., 1997, 3, 917-921). Apoptosis is a process multicellular organisms have evolved to prevent uncontrolled cell proliferation as well as to eliminate cells that have become sick, deleterious, or are no longer necessary. The process of apoptosis involves a multistep cascade in which cells are degraded from within through the concerted action of proteolytic enzymes and DNA endonucleases, resulting in the formation of apoptotic bodies that are then removed by scavenger cells. Research to date has shown that much of the intracellular degradation is carried out through the action of the caspases, a family of proteolytic enzymes that cleave adjacent to aspartate residues (Cohen, Biochemistry Journal, 1997, 326, 1-16).
The finding that most tumor cells display resistance to the apoptotic process has led to the view that therapeutic strategies aimed at attenuating the resistance of tumor cells to apoptosis could represent a novel means to halt the spread of neoplastic cells (Ambrosini et al., Nat. Med., 1997, 3, 917-921). One of the mechanisms through which tumor cells are believed to acquire resistance to apoptosis is by overexpression of survivin, a recently described member of the IAP (inhibitor of apoptosis) caspase inhibitor family. To date, overexpression of survivin has been detected in tumors of the lung, colon, pancreas, prostate, breast, stomach, non-Hodgkin's lymphoma, and neuroblastoma (Adida et al., Lancet, 1998, 351, 882-883; Ambrosini et al., Nat. Med., 1997, 3, 917-921; Lu et al., Cancer Res., 1998, 58, 1808-1812). A more detailed analysis has been performed in neuroblastoma where it was found that survivin overexpression segregated with tumor histologies known to associate with poor prognosis (Adida et al., Lancet, 1998, 351, 882-883). Finally, Ambrosini et al. describe transfection of HeLa cells with an expression vector containing a 708 nt fragment of the human cDNA encoding effector cell protease receptor 1 (EPR-1), the coding sequence of which is complementary to the coding strand of survivin (Ambrosini et al., J. Bio. Chem., 1998, 273, 11177-11182). This construct caused a reduction in cell viability.
Survivin has recently been found to play a role in cell cycle regulation. It has been found to be expressed in the G2/M phase of the cell cycle in a cycle-regulated manner, and associates with microtubules of the mitotic spindle. Disruption of this interaction results in loss of survivin's anti-apoptotic function and increased caspase-3 activity during mitosis. Caspase-3 is associated with apoptotic cell death. It is therefore believed that survivin may counteract a default induction of apoptosis in G2/M phase. It is believed that the overexpression of survivin in cancer may overcome this apoptotic checkpoint, allowing undesired survival and division of cancerous cells. The survivin antisense construct described by Ambrosini above was found to downregulate endogenous survivin in HeLa cells and to increase caspase-3-dependent apoptosis in cells in G2/M phase. Li et al., Nature, 1998, 396, 580-584.
In many species, introduction of double-stranded RNA (dsRNA) induces potent and specific gene silencing. This phenomenon occurs in both plants and animals and has roles in viral defense and transposon silencing mechanisms. (Jorgensen et al., Plant Mol. Biol., 1996, 31, 957-973; Napoli et al., Plant Cell, 1990, 2, 279-289).
The first evidence that dsRNA could lead to gene silencing in animals came from work in the nematode, Caenorhabditis elegans, where it has been shown that feeding, soaking or injecting dsRNA (a mixture of both sense and antisense strands) results in much more efficient silencing than injection of either the sense or the antisense strands alone (Guo and Kemphues, Cell, 1995, 81, 611-620; Fire et al., Nature 391:806-811 (1998); Montgomery et al., Proc. Natl. Acad. Sci. USA 95:15502-15507 (1998); PCT International Publication WO99/32619; (Fire et al., Nature, 1998, 391, 806-810; Timmons et al., Gene, 2001, 263, 103-112; Timmons and Fire, Nature, 1998, 395, 854). Since, the phenonmenon has been demonstrated in a number of organisms, including Drosophila melanogaster (Kennerdell et al., Cell 95:1017-1026 (1998)); and embryonic mice (Wianny et al, Nat. Cell Biol. 2:70-75 (2000)).
This posttranscriptional gene silencing phenomenon has been termed “RNA interference” (RNAi) and has come to generally refer to the process of gene silencing involving dsRNA which leads to the sequence-specific reduction of gene expression via target mRNA degradation (Tuschl et al., Genes Dev., 1999, 13, 3191-3197).
It has been demonstrated that 21- and 22-nt dsRNA fragments having 3′ overhangs are the canonical sequence-specific mediators of RNAi. These fragments, which are termed short interfering RNAs (siRNAs), are generated by an RNase III-like processing reaction from longer dsRNA. Chemically synthesized siRNA also mediate efficient target RNA cleavage with the site of cleavage located near the center of the region spanned by the guiding strand of the siRNA. (Elbashir et al., Nature, 2001, 411, 494-498). Characterization of the suppression of expression of endogenous and heterologous genes caused by the 21-23 nucleotide siRNAs has been investigated in several mammalian cell lines, including human embryonic kidney (293) and HeLa cells (Elbashir et al., Genes and Development, 2001, 15, 188-200).
Recently, it has been shown that single-stranded RNA oligomers (ssRNAi or asRNA) of antisense polarity can be potent inducers of gene silencing and that single-stranded oligomers are ultimately responsible for the RNAi phenomenon (Tijsterman et al., Science, 2002, 295, 694-697).
U.S. Pat. Nos. 5,898,031 and 6,107,094, each of which is herein incorporated by reference, describe certain oligonucleotides having RNA-like properties. When hybridized with RNA, these oligonucleotides serve as substrates for a dsRNase enzyme with resultant cleavage of the RNA by the enzyme (Crooke, 2000; Crooke, 1999).
As a result of these advances in the understanding of apoptosis and the role that survivin expression is believed to play in conferring a growth advantage to a wide variety of tumor cell types, there is a great desire to provide compositions of matter which can modulate the expression of survivin. It is greatly desired to provide methods of diagnosis and detection of nucleic acids encoding survivin in animals. It is also desired to provide methods of diagnosis and treatment of conditions arising from survivin expression. In addition, improved research kits and reagents for detection and study of nucleic acids encoding survivin are desired. Thus, the present invention provides a class of novel inhibitors of survivin, compositions comprising these compounds, and methods of using the compounds.